Title: Crystal structure of UDP‐<i>N</i>‐acetylglucosamine enolpyruvyl transferase from<i>Haemophilus influenzae</i>in complex with UDP‐<i>N</i>‐acetylglucosamine and fosfomycin
Abstract: Proteins: Structure, Function, and BioinformaticsVolume 71, Issue 2 p. 1032-1037 Structure NoteFree Access Crystal structure of UDP-N-acetylglucosamine enolpyruvyl transferase from Haemophilus influenzae in complex with UDP-N-acetylglucosamine and fosfomycin Hye-Jin Yoon, Hye-Jin Yoon Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, KoreaSearch for more papers by this authorSang Jae Lee, Sang Jae Lee Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, KoreaSearch for more papers by this authorBunzo Mikami, Bunzo Mikami Laboratory of Quality Design and Exploitation, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, JapanSearch for more papers by this authorHyun-Ju Park, Hyun-Ju Park College of Pharmacy, Sungkyunkwan University, Suwon, Kyungki-do 440-746, KoreaSearch for more papers by this authorJakyung Yoo, Jakyung Yoo College of Pharmacy, Sungkyunkwan University, Suwon, Kyungki-do 440-746, KoreaSearch for more papers by this authorSe Won Suh, Corresponding Author Se Won Suh [email protected] Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, KoreaDepartment of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea===Search for more papers by this author Hye-Jin Yoon, Hye-Jin Yoon Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, KoreaSearch for more papers by this authorSang Jae Lee, Sang Jae Lee Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, KoreaSearch for more papers by this authorBunzo Mikami, Bunzo Mikami Laboratory of Quality Design and Exploitation, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011, JapanSearch for more papers by this authorHyun-Ju Park, Hyun-Ju Park College of Pharmacy, Sungkyunkwan University, Suwon, Kyungki-do 440-746, KoreaSearch for more papers by this authorJakyung Yoo, Jakyung Yoo College of Pharmacy, Sungkyunkwan University, Suwon, Kyungki-do 440-746, KoreaSearch for more papers by this authorSe Won Suh, Corresponding Author Se Won Suh [email protected] Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, KoreaDepartment of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea===Search for more papers by this author First published: 04 February 2008 https://doi.org/10.1002/prot.21959Citations: 24AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat INTRODUCTION Peptidoglycan (also known as murein) serves a structural role in the bacterial cell wall. It is a polymer consisting of sugars and amino acids that forms a homogeneous layer outside the plasma membrane of eubacteria. Since it is unique to bacterial cells, its synthesis offers an attractive target for discovery of novel antibiotics. The biosynthetic pathway of peptidoglycan is a complex two-stage process. The first stage, which occurs in the cytoplasm, is the formation of the monomeric building block N-acetylglucosamine-N-acetylmuramyl pentapeptide via a six-step process catalyzed by MurA to MurF enzymes.1 The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA; also called MurZ; EC 2.5.1.7) catalyzes the first committed step of peptidoglycan biosynthesis, that is, transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine to form enolpyruvyl-UDP-N-acetylglucosamine.2 It is essential in both gram-negative and gram-positive bacteria such as Escherichia coli and Streptococcus pneumoniae.3, 4 Fosfomycin, a naturally occurring broad-spectrum antibiotic, is the best-known inhibitor of MurA.1 It is an epoxide that specifically inhibits MurA by forming a covalent adduct with a cysteine residue in the active site (Cys115 in both E. coli and Enterobacter cloacae; Cys117 in H. influenzae).5 Fosfomycin is the drug of choice for the treatment of pediatric gastrointestinal infections resulting from Shiga-like toxin-producing E. coli in Japan.1 However, there is a high frequency of fosfomycin resistance and efforts have been made to discover new classes of MurA inhibitors such as the cyclic disulfide (RWJ-3981), the pyrazolopyrimidine (RWJ-11092), and the purine analog (RWJ-140998).6-8 Crystal structures of MurA from E. cloacae and E. coli were reported previously, which revealed that the active site loop containing a highly conserved cysteine residue can adopt different conformations ranging from an open conformation to a closed conformation. The active site loop of ligand-free MurA from E. cloacae was in an open conformation,9 whereas that of MurA from E. coli complexed with UDP-N-acetylglucosamine and fosfomycin was in a closed conformation.10 In view of the flexible nature of the active-site loop, further structural data on MurA enzymes, in particular from human pathogenic bacteria, are essential for structure-based design of MurA inhibitors. To provide such data, we have initiated structure determination of the 424-residue MurA from H. influenzae. Here we report two crystal structures of H. influenzae MurA: a binary complex with the substrate UDP-N-acetylglucosamine at 2.2 Å resolution and a ternary complex with UDP-N-acetylglucosamine and fosfomycin at 2.3 Å resolution. Our binary complex structure is the first structure of MurA bound with the substrate UDP-N-acetylglucosamine and thus our study complements the previous structures. The binary and ternary complex structures of H. influenzae MurA are highly similar to each other, both displaying a half-open conformation for the active site loop. This is in sharp contrast with the closed conformation of the corresponding loop in the E. coli MurA ternary complex with UDP-N-acetylglucosamine and fosfomycin. Our results will be useful in structure-based design of specific inhibitors of MurA for antibacterial discovery. MATERIALS AND METHODS X-ray data collection and structure determination We previously reported overexpression and crystallization of H. influenzae MurA.11 X-ray diffraction data of the ternary complex were recollected at 100 K at the BL-18B experimental station of Photon Factory, Japan, because we had previously experienced some difficulty in flash-freezing the crystals. The crystal of the ternary complex has unit cell parameters of a = 63.7 Å, b = 124.5 Å, c = 126.3 Å in the I222 (or I212121) space group. The binarycomplex was formed inside the E. coli cells even when we did not add UDP-N-acetylglucosamine into the crystallization condition. Since it was difficult to find the cryo conditions for the previously reported crystals of the binary complex,11 we further optimized crystallization conditions and found that the quality of the crystals of the binary complex could be significantly improved by growing them with the reservoir solution of 1.8M ammonium sulfate and 5–10% (v/v) glycerol. X-ray diffraction data of the binary complex were recollected to considerably higher resolution than before11 at 100 K at the BL-4A experimental station of Pohang Light Source, Korea, with the cryo conditions of 25% glycerol and 1.8M ammonium sulfate. The crystal of the binary complex has unit cell parameters of a = 64.0 Å, b = 123.2 Å, c = 127.7 Å in the I222 (or I212121) space group. We could grow the same crystals of the binary complex in the presence of exogenous UDP-N-acetylglucosamine. We also tried crystallographic binding studies of Johnson & Johnson MurA inhibitors (RWJ-110192 and RWJ-3981)6 but were not successful. We could grow crystals in the presence of UDP-N-acetylglucosamine and the pyrazolopyrimidine inhibitor RWJ-110192 under identical conditions as the ternary complex. However, the inhibitor was not present in the active site of the refined structure, probably because a high concentration of ammonium sulfate in the crystallization medium precluded inhibitor binding. We also tried to cocrystallize H. influenzae MurA in the presence of UDP-N-acetylglucosamine and the cyclic disulfide inhibitor RWJ-3981 but could not obtain any crystals. The structure of the ternary complex with UDP-N-acetylglucosamine and fosfomycin was solved by the molecular replacement method using the model of E. coli MurA (PDB code 1UAE).10 The refined model of the ternary complex of H. influenzae MurA was used in turn to solve the structure of the binary complex with UDP-N-acetylglucosamine by the molecular replacement method. Models were refined with the program CNS,12 including the bulk solvent correction. Several rounds of model building, simulated annealing, positional refinement, and individual B-factor refinement were performed. The correct space group was found to be I222 during refinement. Manual model building was done using the program O.13 The structure figures were drawn using the program PyMOL (http://pymol.sourceforge.net). Data deposition The atomic coordinates and structure factors of H. influenzae MurA have been deposited in the Protein Data Bank under accession codes 2RL1 and 2RL2 for the binary and ternary complexes, respectively. RESULTS AND DISCUSSION We have refined both models of the binary and ternary complexes of H. influenzae MurA to reasonable R-values with excellent stereochemistry (Table I). The model of the binary complex accounts for 420 residues (residues 1–420), UDP-N-acetylglucosamine, a sulfate ion, and 346 water molecules. The model of the ternary complex accounts for 420 residues (residues 1–420), UDP-N-acetylglucosamine, fosfomycin, three sulfate ions, and 241 water molecules. Both UDP-N-acetylglucosamine and fosfomycin are well defined by the electron density [Fig. 1(A)]. In both structures, electron density is missing for C-terminal three residues (421–424) of MurA as well as all eight residues belonging to the C-terminal fusion tag (LEHHHHHH), presumably because they are disordered in the crystal. Figure 1Open in figure viewerPowerPoint Ligand electron density and structural comparisons. (A) Electron density of UDP-N-acetylglucosamine (UDP-NAG) (colored in orange, left panel) and fosfomycin (colored in blue, right panel) bound in the active site of the ternary complex of H. influenzae MurA. The 2Fo − Fc map, contoured at 1.0 σ, is superimposed on the refined model. Fosfomycin covalently bound to Cys117 and UDP-N-acetylglucosamine are shown in stick models. (B) Stereo ribbon diagram of superimposed structures of the H. influenzae MurA binary complex (colored in gray) and ternary complex (colored in blue). (C) Stereo ribbon diagram of superimposed MurA structures with a half-open conformation of the active site loop. The loop adopts similar half-open conformations in H. influenzae MurA (blue) complexed with UDP-N-acetylglucosamine and fosfomycin, E. coli MurA (magenta) complexed with a tetrahedral reaction intermediate (1A2N),14 and E. cloacae MurA (cyan) complexed with a reaction product (1RYW).15 UDP-N-acetylglucosamine (orange) and fosfomycin (blue) bound to H. influenzae MurA are shown in stick models. (D) Stereo ribbon diagram of superimposed MurA structures with different conformations of the active site loop. The loop takes a closed conformation in E. coli MurA (green) complexed with UDP-N-acetylglucosamine and fosfomycin (1UAE).10 It adopts a half-open conformation in H. influenzae MurA (blue) complexed with UDP-N-acetylglucosamine and fosfomycin. It has an open conformation in the unliganded E. cloacae MurA (pink) (1NAW).9 UDP-N-acetylglucosamine (orange) and fosfomycin (blue) bound to H. influenzae MurA, as well as fosfomycin bound to E. coli MurA (green), are shown in stick models. Table I. Refinement Statistics Data set Binary complex Ternary complex Resolution range (Å) 30–2.20 20–2.30 Data completeness (%) 96.2 99.6 No. of reflections (working/free set) 22,469/2,563 19,804/2,210 Rwork/Rfreea (%) 19.9/24.8 18.6/23.2 Average B-factors (Å2) Protein atoms (no. residues) 28.7 (420) 29.3 (420) UDP-N-acetylglucosamine 26.5 26.2 Fosfomycin — 48.8 Sulfate ions (number) 28.7 (1) 62.9 (3) Water oxygen atoms (number) 37.8 (346) 33.3 (241) RMS deviations from ideal geometry Bond lengths (Å)/bond angles (°) 0.0057/1.30 0.0054/1.29 a , where Rfree is calculated for a randomly chosen 10% of reflections, which were not used for structure refinement and Rwork is calculated for the remaining reflections. H. influenzae MurA is highly similar in its overall fold to E. coli and E. cloacae enzymes, as one can expect from high levels of amino acid sequence identity among them (72% between H. influenzae and E. coli; 74% between H.influenzae and E. cloacae) [Fig. 2]. The structures of binary and ternary complexes of H. influenzae MurA are highly similar to each other, with a root-mean-square (RMS) deviation of 0.38 Å for 420 Cα atoms [Fig. 1(B)]. When we compare the H. influenzae MurA ternary structure with that of E. coli MurA in complex with UDP-N-acetylglucosamine and fosfomycin (1UAE),10 the RMS deviation is 0.63 Å for 418 Cα atom pairs. However, residues Leu113-Ser118 belonging to the active site loop of H. influenzae MurA shows a large deviation from the corresponding residues of the E. coli MurA structure (1UAE),10 with a maximum Cα deviation of 7.8 Å for Gly116. This residue is N-terminal to the active site Cys117. There is a smaller deviation in the active site loop structure between the binary complex of E. coli MurA bound with a fluorinated reaction intermediate (1A2N)14 and the ternary complex of H. influenzae MurA [Fig. 1(C)]. Between the H. influenzae ternary complex and the E. coli binary complex (1A2N),14 the RMS deviation is 0.63 Å for 418 Cα atom pairs, with a maximum Cα deviation of 3.6 Å for Gly115. Between the H. influenzae binary complex and the E. coli binary complex (1A2N),14 the RMS deviation is 0.68 Å for 418 Cα atom pairs, with a maximum Cα deviation of 4.0 Å for Gly115. These results indicate that the active site loop of H. influenzae MurA undergoes no large conformational change upon covalent linkage of fosfomycin and thus the ternary complex remains in the half-open conformation as the binary complex with UDP-N-acetylglucosamine [Fig. 1(C)]. In contrast, the active site loop of E. coli MurA containing Cys115 is in the closed conformation when it is bound with both UDP-N-acetylglucosamine and fosfomycin (1UAE)10 [Fig. 1(D)]. Figure 2Open in figure viewerPowerPoint Alignment of MurA sequences. The residue numbers are for MurA from H. influenzae. HI is for MurA from H. influenzae (SWISS-PROT entry P45025), EC for E. coli (P0A749), EC2 for E. cloacae (P33038), HP for Helicobacter pylori (P56189), MT for Mycobacterium tuberculosis (P0A5L2), BS for Bacillus subtilis (P19670), and SA for Staphylococcus aureus (P65456). Strictly conserved residues and semiconserved residues are colored in pink and yellow, respectively. The inhibitor fosfomycin binds covalently to a cysteine of the active site loop (indicated by a deep blue box). Two residue insertions in a variable loop between strands 4 and 5 are also indicated by a light blue box. The secondary structures of H. influenzae MurA are indicated above the sequences. This figure was drawn with ClustalX16 and GeneDoc (http://www.nrbsc.org/downloads/). When we compare the H. influenzae MurA ternary structure with the unliganded structure of E. cloacae MurA (1NAW),9 the RMS deviation is 1.3 Å for 418 Cα atom pairs, with Ile119 of H. influenzae MurA showing a maximum Cα deviation of 16.1 Å. The active site loop takes a highly open conformation in the unliganded structure of E. cloacae MurA [Fig. 1(D)]. When we compare the H. influenzae MurA ternary structure with E. cloacae MurA bound with a reaction product (1RYW),15 the RMS deviation is 0.44 Å for 418 Cα atom pairs, with Thr69 of H. influenzae MurA showing a maximum Cα deviation of 5.1 Å. This region between the strands β4 and β5 is highly variable in sequence with a two-residue insertion in H. influenzae MurA [Fig. 2]. Half-open conformations of the active site loops in H. influenzae MurA ternary complex and the E. cloacae MurA product-complex (1RYW)15 are similar to each other [Fig. 1(C)]. We note that the active site loops may be affected by the crystal packing forces to different extents among MurA enzymes from H. influenzae, E. coli, and E. cloacae. In the binary and ternary complexes of H. influenzae, the loop is involved in the crystal packing, whereas the loop is not involved in both closed and half-open states of E. coli MurA (1UAE and 1A2N).10, 14 For E. cloacae MurA, the loop is involved in the crystal packing only in the half-open conformation (1RYW).15 In H. influenzae MurA, the half-open conformation of the active site loop may have been stabilized by the crystal packing forces. Nevertheless, our structures indicate that inhibition of MurA by fosfomycin can occur without a large change in the conformation of the active site loop. In the ternary structures of both H. influenzae MurA and E. coli MurA, the substrate UDP-N-acetylglucosamine is bound in highly similar conformations and fosfomycin is covalently linked by a thioester bond to the SG atom of a conserved cysteine residue in the active site loop (Cys117 of H. influenzae MurA and Cys115 of E. coli MurA) [Fig. 1(D)]. However, when we superimpose the ternary structures H. influenzae MurA and E. coli MurA, two fosfomycin molecules are considerably separated from each other, with a distance of 6.7 Å between their phosphorus atoms. Fosfomycin does not contact the bound UDP-N-acetylglucosamine in H. influenzae MurA, in contrast to the close proximity between fosfomycin and UDP-N-acetylglucosamine in E. coli MurA. This difference in the location of fosfomycin relative to the substrate is a consequence of different conformations of the active site loop. In summary, our structures of MurA from H. influenzae, together with the previously reported structures of MurA enzymes from E. coli and E. cloacae, show that the active-site loop can adopt one of the three major conformations: a fully open conformation, a half-open conformation, and a closed conformation. Our study reveals that fosfomycin can bind to H. influenzae MurA without inducing a large change in the half-open conformation of the binary complex with UDP-N-acetylglucosamine. This finding may have some implications for structure-based design of MurA inhibitors. Acknowledgements We thank Dr. E. Z. Baum at Johnson & Johnson Pharmaceutical Research and Development for providing the MurA inhibitors (RWJ-3981 and RWJ-110192). We thank beamline staffs for assistance during data collection at Photon Factory (BL-18B) and Pohang Light Source (BL-4A). REFERENCES 1 El Zoeiby A,Sanschagrin F,Levesque RC. Structure and function of the Mur enzymes: development of novel inhibitors. 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